This project focuses on the rational redesign of human butyrylcholinesterase (BChE) in order to accelerate cocaine metabolism in human. Enhancing cocaine metabolism by administration of BChE has been recognized as a promising treatment strategy for cocaine abuse. However, the catalytic activity of this plasma enzyme is three orders-of-magnitude lower against the naturally occurring (-)-cocaine than that against the relatively biologically inactive (+)-cocaine isomer. The primary goal of this project in the previous funding cycle was to understand the mechanistic difference between BChE-catalyzed hydrolyses of (-)- cocaine and (+)-cocaine and to test whether a computational approach works or not for rational design of BChE mutants with an improved catalytic efficiency against (-)-cocaine. Progress on the project has revealed the fundamental catalytic pathways for BChE-catalyzed hydrolyses of (-)-cocaine and (+)-cocaine. We have further developed a novel computational design strategy based on transition state simulation, leading to discovery of several BChE mutants with significantly improved catalytic efficiency against (-)-cocaine compared to all BChE mutants reported in literature. Taking advantage of this promising design strategy and protocol, in the next phase of the project we propose an integrated computational-experimental effort to further improve the catalytic efficiency of BChE against (-)-cocaine. The proposed integrated computational- experimental approach will include a large-scale virtual screening of a variety of hypothetical BChE mutants based on the transition-state modeling and simulation, followed by more sophisticated computational evaluation and wet experimental tests. The Specific Aims include: 1. To determine the detailed reaction coordinates and the corresponding free energy profiles for (-)-cocaine hydrolysis catalyzed by the known high-activity mutants of BChE discovered in the previous funding cycle. 2. To design and discover new BChE mutants with further improved catalytic efficiency against (-)-cocaine by using an extended computational design approach based on the transition state modeling and simulation to evaluate a large number of hypothetical BChE mutants, followed by wet experimental tests including site-directed mutagenesis, protein expression, and catalytic activity assay. The long-term objective of this investigation will be to eventually develop an efficient anti-cocaine medication using a high-activity BChE mutant.